Therapeutic effects of bryostatins, bryologs, and other related substances on head trauma-induced memory impairment and brain injury

ABSTRACT

The invention provides for the use of protein kinase activators or boosters of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) or other neurotrophic factors to treat head trauma. Specifically, the present invention provides methods of treating head trauma comprising the steps of identifying a subject having suffered a head trauma and administering to said subject an amount of a pharmaceutical composition comprising a protein kinase C (PKC) activator or 4-methylcatechol acetic acid (MCBA) and a pharmaceutically acceptable carrier effective to treat at least one symptom of head trauma.

This application claims benefit to U.S. Provisional Application Ser. No.60/900,339, filed on Feb. 9, 2007 and U.S. Provisional Application Ser.No. 60/924,662, filed on May 24, 2007, all of which are herebyincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the treatment of head trauma withcompounds that activate protein kinase C (PKC) or boost nerve growthfactor (NGF), brain-derived neurotrophic factor (BDNF) or otherneurotrophic factors.

BACKGROUND OF THE INVENTION A. Head Trauma

Head injury is a trauma to the head, that may or may not include injuryto the brain (see also brain injury). The incidence (number of newcases) of head injury is 300 per 100,000 per year (0.3% of thepopulation), with a mortality of 25 per 100,000 in North America and 9per 100,000 in Britain. Head trauma is a common cause of childhoodhospitalization.

Head injuries include both injuries to the brain and those to otherparts of the head, such as the scalp and skull. Head injuries may beclosed or open. A closed (non-missile) head injury is one in which theskull is not broken. A penetrating head injury occurs when an objectpierces the skull and breaches the dura mater. Brain injuries may bediffuse, occurring over a wide area, or focal, located in a small,specific area. A head injury may cause a skull fracture, which may ormay not be associated with injury to the brain. Some patients may havelinear or depressed skull fractures. If intracranial hemorrhage, orbleeding within the brain occurs, a hematoma within the skull can putpressure on the brain. Types of intracranial hematoma include subdural,subarachnoid, extradural, and intraparenchymal hematoma. Craniotomysurgeries are used in these cases to lessen the pressure by draining offblood. Head trauma is caused by a concussive event.

Brain injury can be at the site of impact, but can also be at theopposite side of the skull due to a contrecoup effect (the impact to thehead can cause the brain to move within the skull, causing the brain toimpact the interior of the skull opposite the head-impact). If theimpact causes the head to move, the injury may be worsened, because thebrain may ricochet inside the skull (causing additional impacts), or thebrain may stay relatively still (due to inertia) but be hit by themoving skull.

B. Protein Kinase C

PKC has been identified as one of the largest gene families ofnon-receptor serine-threonine protein kinases. Since the discovery ofPKC in the early eighties by Nishizuka and coworkers (Kikkawa el al.(1982) J. Biol. Chem. 257: 13341), and its identification as a majorreceptor for phorbol esters (Ashendel et al. (1983) Cancer Res., 43:4333), a multitude of physiological signaling mechanisms have beenascribed to this enzyme. The intense interest in PKC stems from itsunique ability to be activated in vitro by calcium and diacylglycerol(and its phorbol ester mimetics), an effector whose formation is coupledto phospholipid turnover by the action of growth and differentiationfactors.

The activation of PKC has been shown to improve learning and memory.(U.S. patent application Ser. Nos. PCT/US02/13784; PCT/US03/07102;60/287,721; 60/362,081; 10/172,005; and 10/476,459; each incorporatedherein by reference in its entirety). Prior to the present disclosure,however, the PKC-mediated improvement of learning and memory has notbeen recognized as a mechanism for the treatment of post-head traumamemory deficits and brain injury. Also, the PKC activators disclosedherein, specifically those compounds that improve learning and memory,were not recognized as possessing brain function-restoring activityafter head trauma.

Head trauma therapy has historically been limited to few treatmentoptions available. Although many types of potential neuroprotectantshave been tested in clinical trials, none has been approved for clinicaluse, because of ineffectiveness especially when used post-head trauma orassociated toxicity. The compounds presented in this inventiondisclosure were effective when the treatment was started one hour afterthe head trauma in the animal model at doses that have already beendemonstrated to be well tolerated in humans (the bryostatin-1 doses).Compounds that target the protein kinase C (PKC) such as bryostatin-1, adirect PKC activator, and methylcatechol diacetic acid, a derivative ofmethylcatechol, an enhancer of nerve growth factor (NGF), brain-derivedneurotrophic factor (BDNF) or other neurotrophic factors, which isperhaps one of the PKC targets, have been found to have therapeuticvalue against brain injury and memory impairment induced with headtrauma. The development of these substances as therapeutic in thetreatment of head trauma is provided by this invention.

SUMMARY OF THE INVENTION

The present invention provides methods of treating head traumacomprising the steps of identifying a subject having suffered a headtrauma and administering to said subject an amount of a pharmaceuticalcomposition comprising a protein kinase C (PKC) activator or4-methylcatechol acetic acid (MCBA) and a pharmaceutically acceptablecarrier effective to treat at least one symptom of head trauma.

In one embodiment, the PKC activator is FGF-18, a macrocyclic lactone, abenzolactam, a pyrrolidinone, or a combination thereof. In a preferredembodiment, the macrocyclic lactone is a bryostatin or neristatin. Inanother embodiment, the neristatin is neristatin-1. In anotherembodiment, the bryostatin is bryostatin-1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17 or 18. More preferably, the bryostatin isbryostatin-1.

In another preferred embodiment, the pharmaceutical compositioncomprises 4-methylcatechol acetic acid (MCBA), other derivatives ofmethylcatechol, or a brain derived neurotrophic factor. MCBA and otherderivatives of methylcatechol activate or upregulate nerve growth factor(NGF), brain derived neurotrophic factor (BDNF) or other neurotrophicfactors. NGF activates, upregulates or enhances the activity of PKCwhich in turn upregulates, activates or enhances NGF.

In one embodiment, administration of the pharmaceutical compositions ofthe present invention is initiated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14 days of said head trauma. In another embodiment, saidadministration is initiated between 1 and 2 days, 1 and 3 days, 1 and 4days, 1 and 5 or 1 and 7 days of said head trauma. In anotherembodiment, the administration of the pharmaceutical compositions of thepresent invention is initiated within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of said headtrauma. In yet another embodiment, the administration of thepharmaceutical compositions of the present invention is initiatedbetween 1 and 3, 1 and 5, 1 and 10, 1 and 24, 3 and 5, 3 and 10, 3 and24, 5 and 10, 5 and 24, or 10 and 24 hours after said head trauma. Inyet another embodiment, the administration of the pharmaceuticalcompositions of the present invention is initiated after 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24hours after said head trauma. In yet another embodiment, theadministration of the pharmaceutical compositions of the presentinvention is initiated after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or 21 days after said head trauma.

In one embodiment, treatment comprising the administration of thepharmaceutical compositions of the present invention is continued for aduration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 depicts the escape latencies of mice after minimal traumaticbrain injury (TBI) (30 g) followed by 30 ug/kg bryostatin injectedintraperitoneally.

FIG. 2 depicts the escape latencies of mice after minimal traumaticbrain injury (TBI) (30 g):followed by 20 ug/kg bryostatin injectedintraperitoneally.

FIG. 3 depicts memory retention in mice after minimal TBI (30 g)followed by 204/kg bryostatin injected intraperitoneally.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

As used herein, “administration” of a composition includes any route ofadministration, including oral subcutaneous, intraperitoneal, andintramuscular.

As used herein, “an effective amount” is an amount sufficient to reduceone or more symptoms associated with a head trauma.

As used herein, “protein kinase C activator” or “PKC activator” means asubstance that increases the rate of the reaction catalyzed by proteinkinase C by binding to the protein kinase C.

As used herein, the term “subject” means a mammal.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which the active ingredient may be combinedand which, following the combination, can be used to administer theactive ingredient to a subject. As used herein, the term“physiologically acceptable” ester or salt means an ester or salt formof the active ingredient which is compatible with any other ingredientsof the pharmaceutical composition, which is not deleterious to thesubject to which the composition is to be administered.

As used herein, “pharmaceutically acceptable carrier” also includes, butis not limited to, one or more of the following: excipients; surfaceactive agents; dispersing agents; inert diluents; granulating anddisintegrating agents; binding agents; lubricating agents; sweeteningagents; flavoring agents; coloring agents; preservatives;physiologically degradable compositions such as gelatin; aqueousvehicles and solvents; oily vehicles and solvents; suspending agents;dispersing or wetting agents; emulsifying agents, demulcents; buffers;salts; thickening agents; fillers; emulsifying agents; antioxidants;antibiotics; antifungal agents; stabilizing agents; and pharmaceuticallyacceptable polymeric or hydrophobic materials. Other “additionalingredients” which may be included in the pharmaceutical compositions ofthe invention are known in the art and described, for example in Genaro,ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa., which is incorporated herein by reference.

The formulations of the pharmaceutical compositions described herein maybe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, and other mammals.

Despite progress toward the development of new therapeutic agents andavailability of several animal models, there is still a pressing needfor improved animal models for screening

B. Animal Models of Learning and Memory

The area of memory and learning impairment is rich in animal models thatare able to demonstrate different features of memory and learningprocesses. (See, for example, Hollister, L. E., 1990, Pharmacopsychiat.,23, (Suppl II) 33-36). The available animal models of memory loss andimpaired learning involve measuring the ability of animals to remember adiscrete event. These tests include the Morris Water Maze and thepassive avoidance procedure. In the Morris Water Maze, animals areallowed to swim in a tank divided into four quadrants, only one of whichhas a safety platform beneath the water. The platform is removed and theanimals are tested for how long they search the correct quadrant versethe incorrect quadrants. In the passive avoidance procedure the animalremembers the distinctive environment in which a mild electric shock isdelivered and avoids it on a second occasion. A variant of the passiveavoidance procedure makes use of a rodent's preference for dark enclosedenvironments over light open ones. Further discussion can be found inCrawley, J. N., 1981, Pharmacol. Biochem. Behay., 15, 695-699; Costall,B. et al, 1987, Neuropharmacol., 26, 195-200; Costall, B. et al., 1989,Pharmacol. Biochem. Behay., 32, 777-785; Barnes, J. M. et al., 1989, Br.J. Pharmacol., 98 (Suppl) 693P; Barnes, J. M. et al., 1990, Pharmacol.Biochem. Behay., 35, 955-962.

C. Protein Kinase C (PKC)

The PKC gene family consists presently of 11 genes which are dividedinto four subgroups: 1) classical PKCα, β₁, β₂ (β₁ and β₂ arealternatively spliced forms of the same gene) and γ, 2) novel PKCδ, ϵ,η, and θ, 3) atypical PKCζ, λ, η and i and 4) PKC μ. PKC μ resembles thenovel PKC isoforms but differs by having a putative transmembrane domain(reviewed by Blohe et al. (1994) Cancer Metast. Rev. 13: 411; Ilug etal. (1993) Biochem J. 291: 329; Kikkawa et al. (1989) Ann. Rev. Biochem.58: 31). The α, β₁, β₂ and γ isoforms are C²⁺, phospholipid anddiacylglycerol-dependent and represent the classical isoforms of PKC,whereas the other isoforms are activated by phospholipid anddiacylglycerol but are not dependent on Ca²⁺. All isoforms encompass 5variable (V1-V5) regions, and the α, β and γ isoforms contain four(C1-C4) structural domains which are highly conserved. All isoformsexcept PKC α, β and γ lack the C2 domain, the λ η and isoforms also lacknine of two cysteine-rich zinc finger domains in C1 to whichdiacylglycerol binds. The C1 domain also contains the pseudosubstratesequence which is highly conserved among all isoforms, and which servesan autoregulartory function by blocking the substrate-binding site toproduce an inactive conformation of the enzyme (House et al. (1987)Science 238, 1726).

Because of these structural features, diverse PKC isoforms are thoughtto have highly specialized roles in signal transduction in response tophysiological stimuli (Nishizuka (1989) Cancer 10: 1892), as well as inneoplastic transformation and differentiation (Glazer (1994) ProteinKinase C, J. F. Kuo, ed., Oxford U. Press at pages 171-198). For adiscussion of known PKC modulators see PCT/US97/08141, U.S. Pat. Nos.5,652,232; 6,080,784; 5,891,906; 5,962,498; 5,955,501; 5,891,870 and5,962,504 (each incorporated herein by reference in its entirety).

There is increasing evidence that the individual PKC isozymes playdifferent, sometimes opposing, roles in biological processes, providingtwo directions for pharmacological exploitation. One is the design ofspecific (preferably, isozyme specific) inhibitors of PKC. This approachis complicated by the act that the catalytic domain is not the domainprimarily responsible for the isotype specificity of PKC. The otherapproach is to develop isozyme-selective, regulatory site-directed PKCactivators. These may provide a way to override the effect of othersignal transduction pathways with opposite biological effects.Alternatively, by inducing down-regulation of PKC after acuteactivation, PKC activators may cause long term antagonism. Bryostatin iscurrently in clinical trials as an anti-cancer agent. The bryostatinsare known to bind to the regulatory domain of PKC and to activate theenzyme. Bryostatins are examples of isozyme-selective activators of PKC.(see for example WO 97/43268; incorporated herein by reference in itsentirety). For a discussion of known PKC modulators see PCT/US97/08141,U.S. Pat. Nos. 5,652,232; 6,043,270; 6,080,784; 5,891,906; 5,962,498;5,955,501; 5,891,870 and 5,962,504 (each of which is incorporated hereinby reference in its entirety).

Several classes of PKC activators have been identified. Phorbol esters,however, are not suitable compounds for eventual drug developmentbecause of their tumor promotion activity, (Ibarreta et al. (1999) NeuroReport 10 (5&6): 1035-40). Of particular interest are macrocycliclactones (i.e. bryostatin class and neristatin class) that act tostimulate PKC. Of the bryostatin class compounds., bryostatin-1 has beenshown to activate PKC and proven to be devoid of tumor promotionactivity. Bryostatin-1, as a PKC activator, is also particularly usefulsince the dose response curve of bryostatin-1 is biphasic. Additionally,bryostatin-1 demonstrates differential regulation of PKC isozymes,including PKCα, PKCδ and PKCϵ. Bryostatin-1 has undergone toxicity andsafety studies in animals and humans and is actively investigated as ananti-cancer agent. Bryostatin-1's use in the studies has determined thatthe main adverse reaction in humans is myalgia. One example of aneffective dose is 20 or 30 μg/kg per dose by intraperitoneal injection.

Several classes of PKC activators have been identified. Phorbol esters,however, are not suitable compounds for eventual drug developmentbecause of their tumor promotion activity, (Ibarreta et al. (1999) NeuroReport 10 (5&6): 1035-40). Of particular interest are macrocycliclactones (i.e. bryostatin class and neristatin class) that act tostimulate PKC. Of the bryostatin class compounds, bryostatin-1 has beenshown to activate PKC and proven to be devoid of tumor promotionactivity. Bryostatin-1, as a PKC activator, is also particularly usefulsince the dose response curve of bryostatin-1 is biphasic. Additionally,bryostatin-1 demonstrates differential regulation of PKC isozymes,including PKCα, PKCδ and PKCϵ. Bryostatin-1 has undergone toxicity andsafety studies in animals and humans and is actively investigated as ananti-cancer agent. Bryostatin-1's use in the studies has determined thatthe main adverse reaction in humans is myalgia. One example of aneffective dose is 20 or 30 μg/kg per dose by intraperitoneal injection.

Macrocyclic lactones, and particularly bryostatin-1, are described inU.S. Pat. No. 4,560,774 (incorporated herein by reference in itsentirety). Macrocyclic lactones and their derivatives are describedelsewhere in U.S. Pat. Nos. 6,187,568, 6,043,270, 5,393,897, 5,072,004,5,196,447, 4,833,257, and 4,611,066 (each incorporated herein byreference in its entirety). The above patents describe various compoundsand various uses for macrocyclic lactones including their use as ananti-inflammatory or anti-tumor agent. (Szallasi et al. (1994) Journalof Biological Chemistry 269 (3): 2118-24; Zhang et al. (1996) CanerResearch 56: 802-808; Hennings et al. (1987) Carcinogenesis 8 (9):1343-1346; Varterasian et al. (2000) Clinical Cancer Research 6:825-828; Mutter et al. (2000) Bioorganic & Medicinal Chemistry 8:1841-1860) (each incorporated herein by reference in its entirety).

As will also be appreciated by one of ordinary skill in the art,macrocyclic lactone compounds and their derivatives, particularly thebryostatin class, are amenable to combinatorial synthetic techniques andthus libraries of the compounds can be generated to optimize .pharmacological parameters, including, but not limited to efficacy andsafety of the compositions. Additionally, these libraries can be assayedto determine those members that preferably modulate α-secretase and/orPKC.

Combinatorial libraries high throughput screening of natural productsand fermentation broths has resulted in the discovery of several newdrugs. At present, generation and screening of chemical diversity isbeing utilized extensively as a major technique for the discovery oflead compounds, and this is certainly a major fundamental advance in thearea of drug discovery. Additionally, even after a “lead” compound hasbeen identified, combinatorial techniques provide for a valuable toolfor the optimization of desired biological activity. As will be,appreciated, the subject reaction readily lend themselves to thecreation of combinatorial libraries of compounds for the screening ofpharmaceutical, or other biological or medically-related activity ormaterial-related qualities. A combinatorial library for the purposes ofthe present invention is a mixture of chemically related compounds,which may be screened together for a desired property; said librariesmay be in solution or covalently linked to a solid support. Thepreparation of many related compounds in a single reaction greatlyreduces and simplifies the number of screening processes that need to becarried out. Screening for the appropriate biological property may bedone by conventional methods. Thus, the present invention also providesmethods for determining the ability of one or more inventive compoundsto bind to effectively modulate α-secretase and/or PKC.

A variety of techniques are available in the art for generatingcombinatorial libraries described below, but it will be understood thatthe present invention is not intended to be limited by the foregoingexamples and descriptions. (See, for example, Blondelle et al. (1995)Trends Anal. Chem. 14: 83; U.S. Pat. Nos. 5,359,115; 5,362,899;5,288,514: PCT publication WO 94/08051; Chen et al. (1994) JACCS 1 6:2661: Kerr et al. (1993) JACCS 1 1 5:252; PCT publications WO92/10092,WO93/09668; WO91/07087; and WO93/20242; each of which is incorporatedherein by reference). Accordingly, a variety of libraries on the orderof about 16 to 1,000,000 or more diversomers can be synthesized andscreened for a particular activity or property.

Analogs of bryostatin, commonly referred to as bryologs, are oneparticular class of PKC activators that are suitable for use in themethods of the present invention. The following Table summarizesstructural characteristics of several bryologs, demonstrating thatbryologs vary greatly in their affinity for PKC (from 0.25 nM to 10 μM).Structurally, they are all similar. While bryostatin-1 has two pyranrings and one 6-membered cyclic acetal, in most bryologs one of thepyrans of bryostatin-1 is replaced with a second 6-membered acetal ring.This modification reduces the stability of bryologs, relative tobryostatin-1, for example, in both strong acid or base, but has littlesignificance at physiological pH. Bryologs also have a lower molecularweight (ranging from about 600 to 755), as compared to bryostatin-1(988), a property which facilitates transport across the blood-brainbarrier.

PKC Name Affin (nM) MW Description Bryostatin 1.35 988 2 pyran + 1cyclic acetal + macrocycle 1 Analog 1 0.25 737 1 pyran + 2 cyclicacetal + macrocycle Analog 2 6.50 723 1 pyran + 2 cyclic acetal +macrocycle Analog 7a — 642 1 pyran + 2 cyclic acetals + macrocycleAnalog 7b 297 711 1 pyran + 2 cyclic acetals + macrocycle Analog 7c 3.4726 1 pyran + 2 cyclic acetals + macrocycle Analog 7d 10000 745 1pyran + 2 cyclic acetals + macrocycle, acetylated Analog 8 8.3 754 2cyclic acetals + macrocycle Analog 9 10000 599 2 cyclic acetals

Analog 1 (Wender et al. (2004) Curr Drug Discov Technol. 1: 1; Wender etal. (1998) Proc Natl Acad Sci USA 95: 6624; Wender et al. (2002) Am ChemSoc. 124: 13648 (each incorporated herein by reference in theirentireties)) possesses the highest affinity for PKC. This bryolog isabout 100 times more potent than bryostatin-1. Only Analog 1 exhibits ahigher affinity for PKC than bryostatin. Analog 2, which lacks the Aring of bryostatin-1 is the simplest analog that maintains high affinityfor PKC. In addition to the active bryologs, Analog 7d, which isacetylated at position 26, has virtually no affinity for PKC.

B-ring bryologs are also suitable for use in the methods of the presentinvention. These synthetic bryologs have affinities in the low nanomolarrange (Wender et al. (2006) Org Lett. 8: 5299 (incorporated herein byreference in its entirety)). The B-ring bryologs have the advantage ofbeing completely synthetic, and do not require purification from anatural source.

PKC Binding Affinities for B-Ring Bryologs

A third class of suitable bryostatin analogs is the A-ring bryologs.These bryologs have slightly lower affinity for PKC than bryostatin I(6.5, 2.3, and 1.9 nM for bryologs 3, 4, and 5, respectively) but have alower molecular weight.

A number of derivatives of diacylglycerol (DAG) bind to and activateprotein kinase C (Niedel et al. (1983) Proc. Natl. Acad. Sci. USA 80:36; Mori et al. (1982) J. Biochem (Tokyo) 91: 427; Kaibuchi et al.(1983) J. Biol. Chem. 258: 6701). However, DAG and DAG derivatives areof limited value as drugs. Activation of PKC by diacylglycerols istransient, because they are rapidly metabolized by diacylglycerol kinaseand lipase (Bishop et al. (1986) J. Biol. Chem. 261: 6993; Chung et al.(1993) Am. J. Physiol. 265: C927; incorporated herein by reference intheir entireties). The fatty acid substitution determines the strengthof activation. Diacylglycerols having an unsaturated fatty acid are mostactive. The stereoisomeric configuration is also critical. Fatty acidswith a 1,2-sn configuration are active, while 2,3-sn-diacylglycerols and1,3-diacylglycerols do not bind to PKC. Cis-unsaturated fatty acids aresynergistic with diacylglycerols. In one embodiment of the presentinvention, the term “PKC activator” expressly excludes DAG or DAGderivatives, such as phorbol esters.

Isoprenoids are PKC activators suitable for use in the methods of thepresent invention. Farnesyl thiotriazole, for example, is a syntheticisoprenoid that activates PKC with a Kd of 2.5 μM. Farnesylthiotriazole, for example, is equipotent with dioleoylglycerol (Gilbertet al. (1995) Biochemistry 34: 3916; incorporated herein by reference inits entirety), but does not possess hydrolyzable esters of fatty acids.Farnesyl thiotriazole and related compounds represent a stable,persistent PKC activator. Because of its low MW (305.5) and absence ofcharged groups, farnesyl thiotriazole would readily cross theblood-brain barrier.

Octylindolactam V is a non-phorbol protein kinase C activator related toteleocidin. The advantages of octylindolactam V, specifically the(−)-enantiomer, include greater metabolic stability, high potency(Fujiki et al. (1987) Adv. Cancer Res. 49: 223; Collins et al. (1982)Biochem. Biophys. Res. Commun. 104: 1159; each incorporated herein byreference in its entirety) (EC₅₀=29 nM) and low molecular weight thatfacilitates transport across the blood brain barrier.

Gnidimacrin is a daphnane-type diterpene that displays potent antitumoractivity at concentrations of 0.1-1 nM against murine leukemias andsolid tumors. It acts as a PKC activator at a concentration of ≈3 nM inK562 cells, and regulates cell cycle progression at the G1/S phasethrough the suppression of Cdc25A and subsequent inhibition of cyclindependent kinase 2 (Cdk2) (100% inhibition achieved at 5 ng/ml).Gnidimacrin is a heterocyclic natural product similar to bryostatin, butsomewhat smaller (MW=774.9).

Iripallidal is a bicyclic triterpenoid isolated from Iris pallida.Iripallidal displays anti-proliferative activity in a NCI 60 cell linescreen with GI50 (concentration required to inhibit growth by 50%)values from micromolar to nanomolar range. It binds to PKCα with highaffinity (Ki=75.6 nM). It induces phosphorylation of ERK1/2 in aRasGRP3-dependent manner. M.W. 486.7. Iripallidal is only about half thesize of bryostatin and lacks charged groups.

Ingenol [43] is a diterpenoid related to phorbol but possesses much lesstoxicity. It is derived from the milkweed plant Euphorbia peplus.Ingenol 3,20-dibenzoate, for example, competes with [3H]phorboldibutyrate for binding to PKC (Ki for binding=240 nM) (Winkler et al.(1995) J. Org. Chem. 60: 1381; incorporated herein by reference).Ingenol-3-angelate possesses antitumor activity against squamous cellcarcinoma and melanoma when used topically (Ogbourne et al. (2007)Anticancer Drugs. 18: 357; incorporated herein by reference).

Napthalenesulfonamides, includingN-(n-heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) andN-(6-Phenylhexyl)-5-chloro-1-naphthalenesulfonamide, are members ofanother class of PKC activators. SC-10 activates PKC in acalcium-dependent manner, using a mechanism similar to that ofphosphatidylserine (Ito et al. (1986) Biochemistry 25: 4179;incorporated herein by reference). Naphthalenesulfonamides act by adifferent mechanism from bryostatin and would be expected to show asynergistic effect with bryostatin or a member of another class of PKCactivators. Structurally, naphthalenesulfonamides are similar to thecalmodulin (CaM) antagonist W-7, but are reported to have no effect onCaM kinase.

The linoleic acid derivative DCP-LA (2-[(2-pentylcyclopropyl)methyl]cyclopropaneoctanoic acid) is one of the few known isoform-specificactivators of PKC known. DCP-LA selectively activates PKCϵ with amaximal effect at 100 nM. (Kanno et al. (2006) J. Lipid Res. 47: 1146).Like SC-10, DCP-LA interacts with the phosphatidylserine binding site ofPKC, instead of the diacylglycerol binding site.

An alternative approach to activating PKC directly is to increase thelevels of the endogenous activator, diacylglycerol. Diacylglycerolkinase inhibitors such as6-(2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinypethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one(R59022) and[3-[2-4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quinazolinone(R59949) enhance the levels of the endogenous ligand diacylglycerol,thereby producing activation of PKC (Meinhardt et al. (2002) Anti-CancerDrugs 13: 725).

Growth factor activators, such as the 4-methyl catechol derivatives,such as 4-methylcatechol acetic acid (MCBA), that stimulate thesynthesis and/or activation of growth factors such as NGF and BDNF, alsoactivate PKC as well as convergent pathways responsible forsynaptogenesis and/or neuritic branching.

All books, articles, patents or other publications and references arehereby incorporated by reference in their entireties. Reference to anycompound herein includes the racemate as well as the single enantiomers

EXAMPLES

The following Examples serve to further illustrate the present inventionand are not to be construed as limiting its scope in any way.

Example 1 Animal Model of Head Trauma

Minimal traumatic brain injury (TBI) was produced in mice by aconcussive event using a 30 g mass. One hour post trauma, the micereceived 20 or 30 μg bryostatin per kg doses by intraperitonealinjection. The injections were repeated twice weekly for a total of 5treatments. The effects on learning and memory of bryostatin treatmentin treated animals was tested in the Morris Water Maze.

Example 2 Morris Water Maze

In the Morris Water Maze, animals were allowed to swim in a tank dividedinto four quadrants, only one of which had a safety platform beneath thewater. The platform was removed and the animals were tested for how longthey searched the correct quadrant versus the incorrect quadrants. Inthe passive avoidance procedure the animal remembers the distinctiveenvironment in which a mild electric shock is delivered and avoids it ona second occasion.

Example 3 Bryostatin (30 UG/KG) and Treatment of Minimal TBI

Minimal traumatic brain injury (TBI) was produced in mice by aconcussive event using a 30 g mass. One hour post trauma, the micereceived 30 μs bryostatin per kg doses by intraperitoneal injection. Theinjections were repeated twice weekly for a total of 5 treatments. Theescape latencies in a Morris Water Maze of mice treated with bryostatinafter minimal TBI were compared to animals with minimal TBI, controlanimals receiving no TBI or bryostatin, and animals receiving bryostatinonly. The results are shown in FIG. 1.

Example 4 Bryostatin (20 UG/KG) and Treatment of Minimal TBI

Minimal traumatic brain injury (TBI) was produced in mice by aconcussive event using a 30 g mass. One hour post trauma, the micereceived 20 μg bryostatin per kg doses by intraperitoneal injection. Theinjections were repeated twice weekly for a total of 5 treatments. Theescape latencies in a Morris Water Maze of mice treated with bryostatinafter minimal TBI were compared to animals with minimal TBI, controlanimals receiving no TBI or bryostatin, and animals receiving bryostatinonly. The results are shown in FIG. 2. The memory retention of eachtreatment or control group is tabulated in FIG. 3.

1-19. (canceled)
 20. A method of treating post-head trauma memorydeficits resulting from brain injury comprising the steps of identifyinga human subject having suffered a brain injury resulting from headtrauma and administering to said subject an amount of a pharmaceuticalcomposition comprising a protein kinase C (PKC) activator, and apharmaceutically acceptable carrier effective to treat at least onesymptom of head trauma, wherein the PKC activator is bryostatin-1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or neristatin-1,and wherein the administration is initiated within 3 days of the headtrauma and is continued for a duration in a range from about 1 week toabout 12 weeks.
 21. A method of treating post-head trauma memorydeficits resulting from brain injury comprising the steps of identifyinga human subject having suffered a brain injury resulting from headtrauma and administering to said subject an amount of a pharmaceuticalcomposition comprising a protein kinase C (PKC) activator, and apharmaceutically acceptable carrier effective to treat at least onesymptom of head trauma, wherein the PKC activator is bryostatin-1, andwherein the administration is initiated within 3 days of the head traumaand is continued for a duration in a range from about 1 week to about 12weeks.
 22. A method of treating post-head trauma memory deficitsresulting from brain injury comprising the steps of identifying a humansubject having suffered a brain injury resulting from head trauma andadministering to said subject an amount of a pharmaceutical compositioncomprising a protein kinase C (PKC) activator, and a pharmaceuticallyacceptable carrier effective to treat at least one symptom of headtrauma, wherein the PKC activator is bryostatin-2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, or 18, and wherein the administration isinitiated within 3 days of the head trauma and is continued for aduration in a range from about 1 week to about 12 weeks.
 23. The methodof claim 20, wherein the PKC activator is neristatin-1.
 24. The methodof claim 20, wherein the PKC activator has a molecular weight from about600 to 755 and an affinity for PKC from about 0.25 nM to 10 μM.
 25. Themethod of claim 20, wherein said administration is initiated within 1day of said head trauma.
 26. The method of claim 20, wherein saidadministration is initiated within 2 days of said head trauma or 3 daysof head trauma.
 27. The method of claim 20, wherein said administrationis initiated between 1 and 2 days of said head trauma.
 28. The method ofclaim 20, wherein said administration is initiated between 1 and 3 daysof said head trauma.
 29. The method of claim 20, wherein the treatmentis continued for a duration of 1 week.
 30. The method of claim 20,wherein the treatment is continued for a duration of 2 weeks, 3 weeks,or 4 weeks.
 31. The method of claim 20, wherein the treatment iscontinued for a duration of 6 weeks.
 32. The method of claim 21, whereinsaid administration is initiated within 1, 2, or 3 days of said headtrauma.
 33. The method of claim 22, wherein said administration isinitiated within 1, 2, or 3 days of said head trauma.
 34. The method ofclaim 23, wherein said administration is initiated within 1, 2, or 3days of said head trauma.
 35. The method of claim 21, wherein saidadministration is initiated between 1 and 3 days of said head trauma.36. The method of claim 22, wherein said administration is initiatedbetween 1 and 3 days of said head trauma.
 37. The method of claim 21,wherein the treatment is continued for a duration of 1 week, 2 weeks, 3weeks, 4 weeks, or 6 weeks.
 38. The method of claim 22, wherein thetreatment is continued for a duration of 1 week, 2 weeks, 3 weeks, 4weeks, or 6 weeks.
 39. The method of claim 23, wherein the treatment iscontinued for a duration of 1 week, 2 weeks, 3 weeks, 4 weeks, or 6weeks.